Summary of Work: We are examining the relationship between the Xray crystal structures of E. coli DNA polymerase I Klenow fragment and mammalian DNA polymerase beta and their kinetic properties, DNA binding affinity, processivity and fidelity. This year, we completed studies of two Klenow mutators. A flexible 50 amino acid subdomain at the tip of Klenow's thumb interacts with the minor groove of the duplex template- primer. Klenow containing a 24 amino acid deletion that removes a portion of the tip of the thumb has relatively normal dNTP binding and catalytic rate, but reduced DNA binding affinity and lower processivity. The mutant polymerase has relatively normal base substitution fidelity but strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs. The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage. These observations suggest a role for the tip of the thumb in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA. A Y766S polymerase and a Y766A mutant both have elevated base substitution error rates. The magnitude of the mutator effect is mispair specific, from no effect for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. Both the Y766S and Y766A mutant polymerases are also frameshift mutators. Their error specificities suggest that the frameshifts may be initiated by misinsertion rather than slippage. This year we also began a number of studies based on the rich structural information available for beta polymerase. These experiments are intended to further probe several concepts for how DNA polymerases accurately copy DNA. It is our belief that structure function studies of DNA polymerases will improve our understanding of how the human genome is stably replicated and maintained, and how DNA adducts affect gemone stability.